US10688151B2 - Peptides having specificity for the lungs - Google Patents
Peptides having specificity for the lungs Download PDFInfo
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- US10688151B2 US10688151B2 US14/911,197 US201414911197A US10688151B2 US 10688151 B2 US10688151 B2 US 10688151B2 US 201414911197 A US201414911197 A US 201414911197A US 10688151 B2 US10688151 B2 US 10688151B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
Definitions
- the invention relates to a peptide, polypeptide, or protein that binds specifically to cells of the lung endothelium.
- the peptide, polypeptide, or protein can be a component of a viral capsid and can be used to lead a recombinant viral vector selectively to the lung endothelial tissue after systemic administration to a subject and to ensure tissue-specific expression of one or more transgenes there.
- the invention thus further relates to a recombinant viral vector, preferably an AAV vector, which comprises a capsid comprising the peptide, polypeptide, or protein according to the invention and which comprises at least one transgene packaged in the capsid.
- the viral vector is suitable in particular for the therapeutic treatment of a lung disorder or a lung disease.
- the invention further relates to cells and pharmaceutical compositions which comprise the viral vector according to the invention.
- Pulmonary hypertension is a serious chronic lung disease which regularly leads to death if untreated.
- the term applies to diseases of various causes, which are characterized by a structural change in the pulmonary vasculature, and in which there is an increase in blood pressure in the pulmonary arterial system to more than 25 mm Hg [1]. This usually results, in affected patients, in stress-dependent shortness of breath, and general loss of capacity. Disease progression leads to a narrowing of vessels resulting from a transformation (remodeling) and thickening of all three layers of the vessel wall, i.e. intima, media and adventitia [2].
- Pulmonary arterial hypertension is a particularly severe form of pulmonary hypertension in which the median survival from diagnosis is only about three years [3], and diagnosis is often made very late due to the initially mild symptoms.
- Adeno-associated viruses are promising candidates for use in clinical practice because they are classified as relatively safe.
- AAV vectors are able to introduce a transgene into a tissue and express the gene stably and efficiently in the tissue. At the same time, these vectors have no known pathogenic mechanisms [10].
- AAV vectors of serotype 2 AAV2
- the transgenes can be incorporated in different forms in the transfected cells—for example as episomal, single- or double-stranded DNA. Concatamer forms of the DNA have also been demonstrated in transduced cells.
- the genome of AAV2 is formed by a linear, single-stranded DNA molecule of approximately 4700 nucleotides in length and has inverted terminal repeats (ITRs) at both ends.
- the genome also includes two large open reading frames which are called the replication region (rep) and the capsid region (cap).
- the replication region encodes proteins that are required as part of the virus replication.
- the capsid region encodes for the structural proteins VP1, VP2 and VP3, which make up the icosahedral capsid of the virus.
- wild-type AAV vectors such as the AAV2 vectors described above
- systemic administration of wild-type vectors leads to insufficient transduction of lung tissue, and severe immune reactions are expected in the treatment subject due to the unwanted transduction of other tissues.
- peptide ligands which are able to direct the vectors to a particular organ [11-12]. It has been shown that certain peptide ligands bring about a “homing” to various organs such as the brain.
- Reading [13] describes a method which enables the screening for tropism-modified capsids of AAV2 in randomized peptide libraries. From these libraries, vectors can be isolated which specifically transduce a desired cell type in vitro. However, it has been surprisingly found that capsids selected in this manner are often unsuitable for use in vivo because they lack the necessary specificity in animal models [14].
- agents that are able to modulate the tropism of viral vectors and thus ensure adequate cell or tissue specificity to enable targeted delivery of a viral vector into the lung.
- Such vectors enable specific expression of therapeutic genes in lung tissue, for the corresponding, effective treatment of diseases and/or disorders of the lungs.
- the present invention makes available viral vectors for targeted gene transfer to the lungs.
- the viral vectors according to the invention express on their capsid surface a previously unknown amino acid sequence that is specifically recognized in vivo by receptors on the endothelial tissue of the lung.
- the viral vectors of the present invention specifically transduce the lung tissue of a patient following systemic administration to the same.
- the viral vectors according to the invention also enable a strong and persistent expression of a transgene in the endothelial cells of the lung with only minor immune response, and are therefore particularly suitable for gene therapy treatments of certain pulmonary disorders and/or lung diseases. After transfection, the AAV vectors only instigate a minor immune response in the host and are therefore particularly suitable for gene therapy.
- various lung-specific sequences were identified by selection in a randomized AAV2 heptamer peptide library. Beginning with the identified peptide sequence ESGHGYF (SEQ ID NO: 2), the recombinant viral vector rAAV2-ESGHGYF (SEQ ID NO:9) was prepared, in which the peptide sequence ESGHGYF (SEQ ID NO: 2) is expressed as a partial sequence of the capsid protein VP1. Subsequently, the vector rAAV2-ESGHGYF (SEQ ID NO:9) was administered intravenously to mice, and a unique specificity of the vector for lung endothelial tissue was observed both in vitro and in vivo.
- the present invention therefore provides various lung-specific peptide sequences which are particularly suited for directing therapeutic agents such as viral vectors to the lungs of a subject being treated.
- a first aspect of the present invention accordingly relates to a peptide, polypeptide, or protein that binds specifically to endothelial cells in the lungs, wherein the peptide, polypeptide, or protein comprises the amino acid sequence of SEQ ID NO: 1.
- the peptide, polypeptide, or protein preferably comprises the amino acid sequence of SEQ ID NO: 2 or a variant thereof which differs from the amino acid sequence of SEQ ID NO: 2 by the modification of at least one of the two N-terminal amino acids.
- the peptide, polypeptide or protein according to the invention preferably binds to endothelial cells of the lung—in particular, the human lung.
- the invention relates to a peptide, polypeptide or protein which binds specifically to endothelial cells of the lung, wherein the peptide, polypeptide or protein comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5,
- the term “peptide” refers to a linkage of 2-10 amino acids which are connected to each other by a peptide bond.
- the term “polypeptide” refers to a linkage of 11-100 amino acids that are connected to each other by a peptide bond. Polypeptides with more than 100 amino acids are referred to herein as a “protein.”
- the lung-specific peptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or the variant of SEQ ID NO: 2, according to the invention is a part of a capsid protein of a virus. This means that the lung specific sequence is present as part of a capsid protein of the virus.
- a corresponding nucleotide sequence which codes for the lung-specific peptide is cloned into the region of the virus genome which codes for a capsid protein of the virus. If the lung specific sequence is expressed as part of a capsid protein, it can be presented in many copies across the surface of the viral vector.
- the capsid protein is preferably one which is derived from an adeno-associated virus (AAV).
- AAV can be any of the serotypes described in the prior art, wherein the capsid protein is preferably derived from an AAV of one of the serotypes 2, 4, 6, 8 and 9.
- a capsid protein of an AAV of serotype 2 is particularly preferred.
- the capsid of the AAV wild-type is made up of the capsid proteins VP1, VP2 and VP3, which are encoded by the overlapping cap region. All three proteins have the same C-terminal region.
- the capsid of AAV comprises about 60 copies of the proteins VP1, VP2 and VP3, expressed in a ratio of 1:1:8. If a nucleotide sequence coding for one of the lung-specific peptides described herein is cloned at the C-terminus into the reading frame of VP1 (i.e., in the region that is identical in all three proteins), it can be expected that theoretically 60 of the specific peptides can be found on the capsid surface.
- the nucleotide sequence coding for the lung-specific peptide is then cloned into the cap region at the 3′ end of the genome.
- the gene encoding the lung-specific peptide sequence can be cloned into the genomic sequence of one of the capsid proteins VP1, VP2 or VP3.
- the capsid proteins of AAV2 are illustrated by way of example in SEQ ID NO: 6 (VP1), SEQ ID NO: 7 (VP2), and SEQ ID NO: 8 (VP3).
- the gene encoding the lung-specific peptide sequence is cloned into the reading frame of a VP1 gene, preferably in the VP1 gene of AAV2 shown in SEQ ID NO: 6. It should be noted in this case that the insertion of the cloned sequence does not lead to any change of the reading frame, nor to a premature termination to the translation.
- the methods required for the above are readily apparent to a person skilled in the art.
- the inventive lung specific peptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 (or their variants), or of one of the lung-specific peptide sequences of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, is present in inserted form in the region of the amino acids 550-600 of the VP1 protein of AAV2, in particular the protein of SEQ ID NO: 6.
- the lung-specific peptide sequence is present in inserted form in the region of amino acids 560-600, 570-600, 560-590, 570-590 of the VP1 protein.
- amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or their variants adjoin, by way of example directly behind, one of the following amino acids of the VP1 protein, particularly the protein of SEQ ID NO: 6: 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599 or 600.
- amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or its variants follows the amino acid 588 of the VP1 protein of SEQ ID NO: 6, as shown in the examples. It is possible in this case that one or more, and particularly up to 5 (for example, 1, 2, 3, 4 or 5) amino acids which are the result of the cloning are situated between the arginine residue at position 588 and the first amino acid of SEQ ID NO: 1 or SEQ ID NO: 2, or the variant of SEQ ID NO: 2. Likewise, one or more, and particularly up to 5 (i.e., 1, 2, 3, 4, or 5) amino acids can be situated behind the last amino acid of SEQ ID NO: 1 or SEQ ID NO: 2, or the variant of SEQ ID NO: 2.
- the sites and regions in the amino acid sequence of the capsid protein indicated above for VP1 apply analogously to the capsid proteins VP2 and VP3 of AAV2. Because the three capsid proteins VP1, VP2 and VP3 of AAV2 differ only by the length of the N-terminal sequence and accordingly have an identical C-terminus, a person skilled in the art will have no problem making a sequence comparison to identify the sites indicated above, for the insertion of the peptide ligands, in the amino acid sequences of VP1 and VP2. As such, the amino acid 588 in VP1 corresponds to position R451 of VP2 (SEQ ID NO: 7) and/or position R386 of VP3 (SEQ ID NO: 8).
- SEQ ID NO: 9 shows an example of the sequence of the VP1 protein of AAV2 after introduction of the peptide sequence of SEQ ID NO: 2. Due to the cloning, the capsid protein has two additional amino acids which do not occur in the native sequence of the VP1 protein of AAV2. As such, the peptide sequence of SEQ ID NO: 2 is flanked at its N-terminus by a glycine in position 589, and at its C-terminus by an alanine in position 597. In addition, the asparagine at position 587 of the native sequence is replaced with a glutamine.
- the present invention therefore also relates to a capsid protein which comprises or consists of:
- the invention is directed to a viral capsid, which comprises a peptide, polypeptide or protein which specifically binds to cells of the lung, and which has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant of SEQ ID NO: 2 as described above (or alternatively, the peptide sequences of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5).
- the present invention also provides a nucleic acid encoding a peptide, polypeptide or protein as described above.
- a nucleic acid which encodes a capsid protein which comprises a peptide, polypeptide or protein according to the invention as described above, is likewise provided.
- the nucleic acid coding for the capsid protein comprises the nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence derived from the same, with at least 80% sequence identity.
- a plasmid comprising such a nucleic acid is also provided.
- the invention relates to a recombinant—i.e., produced by means of genetic engineering techniques—viral vector, having a capsid and at least one transgene packaged therein, wherein the capsid comprises at least one capsid protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or of a variant of SEQ ID NO: 2 which differs from the amino acid sequence of SEQ ID NO: 2 by modification of at least one of the two N-terminal amino acids.
- the capsid protein can also comprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
- the recombinant viral vector can be a recombinant AAV vector—for example, of serotype 2, 4, 6, 8, 9. AAV vectors of serotype 2 are particularly preferred.
- the different AAV serotypes differ mainly by their natural tropism. As such, wild-type AAV2 binds more readily to alveolar cells than to lung epithelial cells, while AAV5, AAV6 and AAV9 are capable of greater transduction of epithelial cells of the respiratory tract. A person skilled in the art can take advantage of these natural differences in the specificity of the cells to further amplify the specificity mediated by the peptides according to the invention for certain cells or tissues.
- the various AAV serotypes are highly homologous. For example, serotypes AAV1, AAV2, AAV3 and AAV6 are 82% identical on the nucleic acid level [25].
- the capsid of the viral vectors according to the invention comprises one or more transgenes.
- a gene which has been introduced by genetic engineering into the genome of the vector is termed a transgene.
- the transgene can be DNA or RNA.
- it is single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), such as genomic DNA or cDNA.
- the transgene which is to be transported with the aid of the recombinant viral vector according to the invention is a human gene.
- Suitable transgenes include, for example, therapeutic genes to replace a dysfunctional gene in patients. They can also be genes which are not expressed in the corresponding target tissue, or are only expressed to an insufficient extent.
- the transgene which is expressed from the viral vector according to the invention is a gene encoding a nitric oxide synthase (NOS) or the bone morphogenic protein receptor 2 (BMPR2).
- NOS nitric oxide synthase
- BMPR2 bone morphogenic protein receptor 2
- eNOS endothelial NOS
- iNOS inducible NOS
- the genes encoding eNOS and iNOS can be used for the treatment of a pulmonary disorder or pulmonary disease in a patient—in particular pulmonary hypertension or pulmonary arterial hypertension.
- the transgene encodes a radioprotective protein such as a radioprotective enzyme.
- a radioprotective protein such as a radioprotective enzyme.
- the lung is a therapeutically particularly relevant site of tumor growth but at the same time a particularly radiosensitive organ, which is why primary tumors there often can only be treated to a limited extent with radiotherapy, and diffuse tumor growth usually cannot be treated at all with radiotherapy.
- lung-specific vectors of the present invention the healthy tissue surrounding the malignant tissue can be protected by targeted expression of radioprotective proteins.
- the transgene which is introduced into the healthy lung tissue is a manganese superoxide dismutase (MnSOD) which catalyzes the conversion of superoxide anions—one of the critical factors in radiation-induced toxicity—to hydrogen peroxide.
- MnSOD manganese superoxide dismutase
- a further embodiment proposed here involves the kinase domains of the ataxia telangiectasia mutant (ATM) gene, which contributes to the repair of DNA damage caused by radiation.
- both genes are introduced by means of the presently described vectors into the patient undergoing treatment.
- the transgene encodes for human alpha-1-antitrypsin.
- the lack of sufficient quantities of alpha-1-antitrypsin is the cause of Laurell-Eriksson syndrome, a hereditary metabolic disease which can lead to emphysema or cirrhosis.
- Alpha-1-antitrypsin is required for the regulation of the activity of proteases in the serum. The lack of this inhibitor leads to increased proteolysis in the serum and accordingly to the severe sequelae named above.
- the viral vectors of the present invention are particularly suitable for use in a method for therapeutic treatment of diseases of the lung.
- Lung disorders in the context of the invention include, in particular, all kinds of vascular diseases of the lung such as pulmonary hypertension, as well as lung tumors, alpha-1-antitrypsin deficiency (A1AD), and others.
- lung tumors which are suitable for treatment using the vectors according to the invention include small cell lung cancer (SCLC), squamous-cell carcinoma, adenocarcinoma, and large cell lung carcinoma.
- the viral vectors of the present invention are used for therapeutic treatment of pulmonary hypertension or pulmonary arterial hypertension.
- the transgene encodes an antitumor agent, such as a tumor suppressor protein, or an immunomodulator such as a cytokine (such as interleukin 1 to 4, gamma-interferon, p53), which is intended to be transported selectively to the lung tissue of the patient.
- an antitumor agent such as a tumor suppressor protein
- an immunomodulator such as a cytokine (such as interleukin 1 to 4, gamma-interferon, p53), which is intended to be transported selectively to the lung tissue of the patient.
- cytokine such as interleukin 1 to 4, gamma-interferon, p53
- vectors can also be used to transport antisense-RNA, ribozymes, or the like into the endothelial tissue of the lung.
- vectors according to the invention can also comprise transgenes encoding secretory proteins that are intended for systemic administration in the bloodstream. Such secretory proteins can be efficiently deposited in the bloodstream via the pulmonary capillary bed, which is part of the cardiovascular system.
- the term “subject” indicates any human or animal organism that can be infected by AAV vectors.
- the subject being treated is a mammal such as a human, a primate, a mouse or a rat.
- the subject to be treated is a human.
- the vector brings about a site-specific expression of the transgene in the cells of the lung endothelium.
- the transgene can be present in the viral vector in the form of an expression cassette, which in addition to the sequence of the transgene to be expressed comprises further elements necessary for expression, such as a suitable promoter which controls the expression of the transgene after infection of the appropriate cells.
- suitable promoters include, in addition to the AAV promoters such as the cytomegalovirus (CMV) promoter or the chicken beta actin/cytomegalovirus hybrid promoter (CAG), an endothelial cell-specific promoter such as the VE-cadherin promoter, as well as steroid promoters and metallothionein promoters.
- the transgene according to the invention comprises a pulmonary endothelium-specific promoter which is connected by a functional bond to the transgene to be expressed.
- a pulmonary endothelium-specific promoter is a promoter whose activity in lung endothelial cells is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold higher than in a cell which is not a pulmonary endothelium cell.
- this promoter is a human promoter.
- the expression cassette can also include an enhancer element for increasing the expression levels of exogenous protein to be expressed.
- the expression cassette can include polyadenylation sequences, such as the SV40 polyadenylation sequences or polyadenylation sequences of bovine growth hormone.
- the viral vectors according to the invention can, preferably as part of one of their capsid proteins, comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
- variants of the amino acid sequence of SEQ ID NO: 2 can be used, the same differing from the amino acid sequence of SEQ ID NO: 2 by the modification of at least one of the two N-terminal amino acids.
- the modification can be a substitution, deletion or insertion of amino acids, as long as the variant retains the ability to communicate, as part of the capsid, the specific binding of the vector to the receptor structures of endothelial cells of the lung.
- the invention therefore also extends to variants of the sequence of SEQ ID NO: 2 in which one of the two N-terminal amino acids of SEQ ID NO: 2 has been changed.
- variants which are within the scope of the invention, therefore have a sequence identity of more than 85% to the amino acid sequence shown in SEQ ID NO: 2 when the sequences are compared using the programs GAP or BESTFIT.
- GAP or BESTFIT programs for determining amino acid sequence identity are sufficiently known in the art.
- the variant of the sequence of SEQ ID NO: 2 can be based on the substitution of one or two of the N-terminal amino acids—that is, one or both N-terminal amino acids can be replaced with another amino acid.
- the substitution by which the variants of the amino acid sequence in SEQ ID NO: 2 differ is a conservative substitution—i.e., a substitution of one amino acid by an amino acid of similar polarity which gives the peptide similar functional properties.
- the substituted amino acid is from the same group of amino acids as the amino acids which are replaced. For example, a hydrophobic residue can be replaced with another hydrophobic residue, or a polar residue by another polar residue.
- Functionally similar amino acids which can be exchanged for each other by a conservative substitution include, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.
- non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan.
- uncharged polar amino acids are serine, threonine, glutamine, asparagine, tyrosine, and cysteine.
- charged, polar (acidic) amino acids include histidine, arginine and lysine.
- charged, polar (basic) amino acids include aspartic acid and glutamic acid.
- amino acid sequences in which an amino acid has been inserted into the region of the two N-terminal amino acids of SEQ ID NO: 2 are also considered variants of the amino acid sequence shown in SEQ ID NO: 2. Such insertions can in principle be carried out as long as the resulting variant retains its ability to bind specifically to endothelial cells of the lung.
- those proteins in which one of the two N-terminal amino acids of SEQ ID NO: 2 is missing are considered to be variants of the amino acid sequence shown in SEQ ID NO: 2. This requires in turn that the correspondingly deleted variant binds specifically to endothelial cells of the lung.
- these modified amino acids can be amino acids that have been modified by biotinylation, phosphorylation, glycosylation, acetylation, branching and/or cyclization.
- Viral vectors with capsids which comprise one of the peptide sequences according to the invention or comprise a variant thereof as defined above bind specifically to endothelial cells of the lung.
- a “specific” binding of the vectors according to the invention means that the vectors accumulate after systemic administration mainly on or in the endothelial cells of the lung. This means that more than 50% of the originally administered vector genomes accumulate in the endothelial cells, or in the area of the endothelial cells, of the lungs, while less than 50% accumulate in other cells or tissues (such as in the spleen or liver), or the area thereof.
- more than 60%, 70%, 80%, 90%, 95%, or even more than 99% of the originally administered vector genomes accumulate in, or in the region of, the endothelial cells of the lung.
- Specific binding of the vectors can also be determined via the expression of the transgene.
- more than 50% of the total expression of the transgene occurs in, or in the region of, the endothelial cells of the lung, while less than 50% of the expression can be observed in, or in the region of, other tissues. It is preferred, however, that more than 60%, 70%, 80%, 90%, 95%, or even more than 99% of the total measured expression of the transgene occurs in, or in the region of, the endothelial cells of the lungs.
- vectors whose capsids comprise variants of the amino acid sequence shown in SEQ ID NO: 2 have at least about 50% of the binding activity of a corresponding viral vector with a capsid which has the amino acid sequence shown in SEQ ID NO: 2. It is even more preferred that the variants have about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding activity of a corresponding viral vector whose capsid has the amino acid sequence shown in SEQ ID NO: 2.
- the binding activity of the vectors can be measured with the aid of in vitro assays, as described in the included examples.
- the binding activity of the vectors according to the invention is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000 fold, 5000-fold, or 10,000-fold higher than the binding activity for a control cell which is not a lung endothelium cell (such as a spleen or liver cell).
- a control cell which is not a lung endothelium cell (such as a spleen or liver cell).
- the invention also relates to a cell that comprises a peptide, polypeptide or protein according to the invention, a nucleic acid encoding the same, a plasmid comprising such a nucleic acid, or a recombinant AAV vector as described above. It is preferably a human cell or cell line.
- a cell has been, for example, obtained from a human subject by biopsy and then transfected with the viral vector in an ex vivo procedure.
- the cell can then be re-implanted in the subject, or be supplied in other ways to the subject—for example by transplantation or infusion.
- the likelihood of rejection of transplanted cells is lower when the subject from which the cell was derived is genetically similar to the subject to which the cell is administered.
- the subject to whom the transfected cells are supplied is the same subject from which the cells were previously obtained.
- the cell is preferably a human lung cell, particularly a cell of the human pulmonary endothelium.
- the cell to be transfected can also be a stem cell, such as a human adult stem cell.
- the cells to be transfected are autologous cells that have been transfected ex vivo with the viral vector according to the invention, for example the recombinant AAV2 vector described above.
- the cells are preferably used in a method for treating a pulmonary disorder or a lung disease in a subject.
- the invention in another aspect, relates to a method for producing an AAV vector in which a plasmid is used which encodes a capsid protein, the same comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant thereof.
- the vector can also comprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
- the basic method of producing recombinant AAV vectors comprising a transgene to be expressed is described in the prior art sufficiently [28].
- HEK 293-T cells are transfected with three plasmids.
- a first plasmid comprises the cap and rep regions of the AAV genome, but the naturally occurring inverted repeats (ITRs) are missing.
- the cap region of this plasmid comprises a gene encoding at least one modified capsid protein, i.e., a gene which comprises the peptide sequence according to the invention.
- a second plasmid comprises a transgene expression cassette which is flanked by the corresponding ITRs, which constitute the packaging signal. The expression cassette is therefore packaged into the capsid in the course of the assembly of the viral particles.
- the third plasmid is an adenoviral helper plasmid, on which are encoded the helper proteins E1A, E1B, E2A, E4orf6, VA, which are required for AAV replication in the HEK 293-T cells.
- the vectors according to the invention can be purified, for example, by gel filtration processes—for example using a Sepharose—desalinated, and subsequently purified by filtration.
- Other purification methods can further comprise a cesium chloride or iodixanol gradient ultracentrifugation process. Purification reduces potentially detrimental effects in the subject to which the adeno-associated viral vectors are administered.
- the administered virus is substantially free of wild-type and replication-competent virus. The purity of the virus can be checked by suitable methods such as PCR amplification.
- the invention provides a pharmaceutical composition comprising a viral vector of the present invention, particularly an AAV vector.
- the viral vector in this case is administered in a therapeutically effective amount to the patient, i.e., in an amount sufficient to considerably improve at least one symptom of the lung dysfunction or lung disease being treated, or to prevent the progression of the disease.
- Symptoms that are regularly associated with a lung disease include cough, fever, chest pain, hoarseness and difficulty breathing.
- a therapeutically effective amount of the vector according to the invention causes a positive change in one of the mentioned symptoms, i.e., a change which results in the phenotype of the affected subject approximating the phenotype of a healthy subject who does not suffer from a pulmonary disease.
- the administration of the viral vector occurs in an amount which leads to a complete or substantially complete healing of the lung dysfunction or lung disease.
- the pharmaceutical composition accordingly comprises a therapeutically effective dose of the vector according to the invention.
- a therapeutically effective dose will generally be non-toxic for the subject who undergoes the treatment.
- the exact amount of viral vector which must be administered to achieve a therapeutic effect depends on several parameters. Factors that are relevant to the amount of viral vector to be administered are, for example, the route of administration of the viral vector, the nature and severity of the lung disease, the disease history of the patient being treated, and the age, weight, height, and health of the patient to be treated. Furthermore, the expression level of the transgene which is required to achieve a therapeutic effect, the immune response of the patient, as well as the stability of the gene product are relevant for the amount to be administered. A therapeutically effective amount of the viral vector can be determined by a person skilled in the art on the basis of general knowledge and the present disclosure.
- the viral vector is preferably administered in an amount corresponding to a dose of virus in the range of 1.0 ⁇ 10 10 to 1.0 ⁇ 10 14 vg/kg (virus genomes per kg body weight), although a range of 1.0 ⁇ 10 11 to 1.0 ⁇ 10 13 vg/kg is more preferred, and a range of 5.0 ⁇ 10 11 to 5.0 ⁇ 10 12 vg/kg is still more preferred, and a range of 1.0 ⁇ 10 12 to 5.0 ⁇ 10 12 is still more preferred.
- a virus dose of approximately 2.5 ⁇ 10 12 vg/kg is most preferred.
- the amount of the viral vector to be administered can be adjusted according to the strength of the expression of one or more transgenes.
- the viral vector of the present invention can be formulated for various routes of administration—for example, for oral administration as a capsule, a liquid or the like.
- the viral vector is administered parenterally, preferably by intravenous injection or intravenous infusion.
- the administration can be, for example, by intravenous infusion, for example within 60 minutes, within 30 minutes or within 15 minutes.
- the viral vector is administered locally by injection to the lung during a surgery.
- Compositions which are suitable for administration by injection and/or infusion typically include solutions and dispersions, and powders from which corresponding solutions and dispersions can be prepared. Such compositions will comprise the viral vector and at least one suitable pharmaceutically acceptable carrier.
- Suitable pharmaceutically acceptable carriers for intravenous administration include bacteriostatic water, Ringer's solution, physiological saline, phosphate buffered saline (PBS) and Cremophor ELTM.
- Sterile compositions for the injection and/or infusion can be prepared by introducing the viral vector in the required amount into an appropriate carrier, and then sterilizing by filtration.
- Compositions for administration by injection or infusion should remain stable under storage conditions after their preparation over an extended period of time.
- the compositions can contain a preservative for this purpose. Suitable preservatives include chlorobutanol, phenol, ascorbic acid and thimerosal.
- the preparation of corresponding formulations and suitable adjuvants is described, for example, in “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins; 21st edition (2005).
- the invention relates to a method for the therapeutic treatment of a pulmonary disorder or a pulmonary disease, wherein a viral vector according to the invention, preferably an AAV vector as described above, is administered to a subject.
- the vector comprises a capsid which has at least one capsid protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variant as described above of SEQ ID NO: 2.
- the vector can also comprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
- the viral vector further comprises a transgene, for example a therapeutic gene, which is useful for the treatment of the pulmonary disorder or the lung disease.
- the vector brings about the specific expression of the gene in the endothelial cells of the lung.
- FIG. 1 shows the in vivo selection method of the AAV-peptide library used according to the invention.
- FIG. 2 shows the sequences of the lung-specific peptides identified by means of the selection method. After the fourth round of selection, a total of four different sequences were identified.
- FIG. 3 shows the measurement of the expression of luciferase 14 days after systemic administration of recombinant AAV vectors in mouse organ lysates.
- A wild-type AAV2 vector (upper panel) and the insertion control AAV2-CVGSPCG (SEQ ID NO:21, middle panel) induce mainly heart-specific expression.
- AAV2-ESGHYGF (SEQ ID NO:9, lower panel) induces a strong expression of luciferase, which is simultaneously lung-specific.
- B Comparison of the expression levels of wild-type AAV2, AAV2-CVGSPCG (SEQ ID NO:21) and AAV2-ESGHYGF (SEQ ID NO:9) vectors in the heart (upper panel), liver (middle panel) and lung (lower panel).
- FIG. 5 shows the distribution of recombinant AAV vectors after systemic administration of 5 ⁇ 10 10 gp/mouse by quantitative real-time PCR.
- A Distribution of AAV2-ESGHYGF (SEQ ID NO:9) 4 hours after vector administration in seven different organs.
- B Distribution of genomes provided by the wild-type AAV2 vector (upper panel), the control vector AAV2-CVGSPCG (SEQ ID NO:21, middle panel) and AAV2-ESGHYGF (SEQ ID NO:9) lower panel).
- the control vector and wild-type vector accumulate in the reticuloendothelial system of the liver and spleen.
- AAV2-ESGHYGF accumulates exclusively in the lungs.
- a random-display peptide library was prepared and selected in four rounds.
- a random X 7 -AAV peptide library with a theoretical diversity of 1 ⁇ 10 8 individually occurring clones was prepared using a two-stage protocol as previously described [26-27].
- a degenerate oligonucleotide was first produced which codes for seven randomized amino acids at nucleotide position 3967 in the AAV genome, which corresponds to the amino acid position R588 in VP1.
- the oligonucleotide had the sequence: 5′-CAGTCGGCCAGAGAGGC(NNK) 7 GCCCAGGCGGCTGACGAG-3′ (SEQ ID NO: 11).
- the second strand was produced using a Sequenase (Amersham, Freiburg, Germany) and the primer with the sequence 5′-CTCGTCAGCCGCCTGG-3′ (SEQ ID NO: 12).
- the double-stranded insert was cut with BglI, purified with the QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany) and ligated into the library with SfiI digested library plasmid pMT187-0-3 [26].
- the diversity of the plasmid library was determined by the number of clones grown from a representative aliquot of transformed, electrocompetent DH5 ⁇ bacteria on agar containing 150 mg/ml ampicillin.
- Library plasmids were harvested and purified by using the Plasmid Preparation Kit from Qiagen.
- the AAV library genomes were packaged into chimeric wild-type and library AAV capsids (AAV transfer shuttle) by transfecting 2 ⁇ 10 8 293T cells in 10 cell culture dishes (15 cm) with the plasmid pVP3 cm (containing the wild-type cap genes with modified codon usage without the inverted terminal repeats) [27], the library plasmids and the pXX6 helper plasmid [28], wherein the ratio between the plasmids was 1:1:2.
- the resulting AAV library transfer shuttles were used to infect 2 ⁇ 10 8 293T cells in cell culture dishes (15 cm) with an MOI of 0.5 replication units per cell.
- the random oligonucleotides that were included in AAV particles of the library and had accumulated in the tissue of interest were amplified by nested PCR using the primers 5′-ATGGCAAGCCACAAGGACGATG-3′ (SEQ ID NO: 15) and 5′-CGTGGAGTACTGTGTGATGAAG-3′ (SEQ ID NO: 16) for the first PCR and the primers 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ (SEQ ID NO: 17) and 5-TGATGAGAATCTGTGGAGGAG-3′ (SEQ ID NO: 18) for the second PCR.
- the PCR-amplified oligonucleotides were used to prepare secondary libraries for three additional rounds of selection.
- the secondary libraries were generated like the primary libraries (see above), but without the additional step of producing transfer shuttles.
- the secondary plasmid library was used to transfect 2 ⁇ 10 8 293T cells in cell culture dishes (15 cm) at a ratio of 25 library plasmids per cell, wherein the transfection reagent Polyfect (Qiagen) was used. After each round of selection, several clones were sequenced. The applied selection method is shown in FIG. 1 .
- the clones enriched in Example 1 were produced as recombinant AAV vectors and tested for their transduction profile.
- Recombinant AAV vectors were produced by triple transfection of HEK293T cells. The cells were incubated at 37° C., 5% CO 2 in Dulbecco's modified Eagle Medium (Invitrogen, Carlsbad, USA), supplemented with 1% penicillin/streptomycin and 10% fetal calf serum. Plasmid DNA was transfected into 293T cells with the transfection agent Polyfect (Qiagen, Hilden, Germany).
- pXX6 was used as adenoviral helper plasmid [28], which encodes the luciferase gene pUF2-CMV-luc [27] or the GFP gene pTR-CMV-GFP [30], as was a plasmid encoding the AAV capsid of interest.
- oligonucleotide inserts were made which encode modified variants of the peptide ESGHGYF (SEQ ID NO: 2).
- the inserts were processed as described into library inserts (see above).
- the genomic titer was determined by the LightCycler system, as previously described [32], by real-time PCR using the CMV-specific primers 5′-GGCGGAGTTGTTACGACAT-3′ (SEQ ID NO: 19) and 5′-GGGACTTTCCCTACTTGGCA-3′ (SEQ ID NO: 20).
- the peptides were introduced into the capsid of a recombinant vector comprising a luciferase reporter gene.
- Vectors with mutated capsids were injected into mice along with control vectors.
- the luciferase expression was analyzed using a Xenogen IVIS200 Imaging System (Caliper Lifescience, Hopkinton, USA) with the Living Image 4.0 (Caliper) software, following intraperitoneal injection of 200 ⁇ l of luciferin substrate (150 mg/kg, Xenogen) per mouse. Representative, in vivo bioluminescence images of the expression of the transgene at different positions (ventral, dorsal, lateral) were taken when the luminescence in relative light units (photons/sec/cm2) reached the highest intensity. Then the animals were sacrificed, the organs of interest were removed quickly, and images of the expression of the transgene in individual organs were immediately taken.
- the organs were then frozen in liquid nitrogen and stored at ⁇ 80° C. Three-dimensional reconstructions of the in vivo luminescence images were obtained by using the DLIT option of the Living Image 4 software, and the emitted light was measured in 5 different wavelengths from 560-640 nm for three minutes each. To quantify the luciferase expression, the organs were homogenized in reporter lysis buffer (RLB, Promega, Madison, USA).
- the determination of the luciferase reporter gene activity was carried out in a luminometer (Mithras LB9 40, Berthold Technologies, Bad Wildbad, Germany) at 10-second intervals after the addition of 100 ⁇ L luciferase assay reagent (LAR, Promega), with a 2-second delay between each of the measurements. The values were normalized in each sample with respect to the total amount of protein using the Roti NanoQuant protein assay (Roth, Düsseldorf, Germany).
- a randomly selected control clone of the non-selected library led to a weak gene expression that occurred primarily in the heart and in some parts of the abdomen, but not in the lung. Wild-type AAV2 caused a weak gene expression in the heart, liver and skeletal muscle, but not in the lungs.
- a three-dimensional reconstruction of bioluminescence images confirmed the lung-specific expression.
- the peptide ADGVMWL (SEQ ID NO: 3), which was also enriched during the in vivo selection, also led to a lung-specific expression of the transgene, but was weaker than for the peptide ESGHGYF(SEQ ID NO: 2).
- the control peptide CVSGPCG (SEQ ID NO:21) produced a moderate gene expression in the heart (8.7 ⁇ 10 4 RLU/mg protein, see FIG. 3A , middle panel).
- vectors which had the lung specific ESGHGYF (SEQ ID NO:2) capsid led to a strong and specific gene expression in the lung (4.1 ⁇ 10 5 RLU/mg protein, see FIG. 3A , lower panel).
- the lung-specific ESGHGYF (SEQ ID NO:2) vectors showed only an expression on the order of the background signal (about 1 ⁇ 10 3 RLU/mg protein).
- the expression of the transgene in the lungs for the ESGHGYF (SEQ ID NO:2) vectors was more than 200-fold higher than the expression mediated by wild-type AAV2 or by the CVGSPCG (SEQ ID NO:21) control vectors (see FIG. 3B ).
- the lung-specific expression of the transgene mediated by the ESGHGYF (SEQ ID NO:2) vectors remained organ-specific over a long period.
- the expression of the transgene was measured over a period of 164 days. The radiation emitted in the lung region was determined quantitatively. Over the entire period of time, the expression of the transgene was stable at a high level, and was limited to the lung. The lowest expression in the lung was measured at day 7, a peak was reached on day 42, and the radiation declined only slowly to the last measurement on day 164 ( FIG. 4 ).
- ESGHGYF ESGHGYF
- the distribution of vectors was investigated four hours after intravenous administration of 5 ⁇ 10 10 gp/mouse.
- the quantification of the vector genomes was performed by real-time PCR.
- the total DNA was extracted from the organ concerned at various time points after intravenous administration of 5 ⁇ 10 10 vg/mouse using a tissue homogenizer (Precellys 24, Peqlab, Er Weg, Germany) and the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
- the DNA was quantified using a spectrophotometer (NanoDrop ND-2000C, Peqlab).
- the analysis of the AAV vector DNA in the tissues was performed by quantitative real-time PCR using the above-described CMV-specific primer, wherein 40 ng of template were used, normalized with respect to the total DNA.
- the quantification of the vector genomes by real-time PCR showed a lung-specific homing of ESGHGYF (SEQ ID NO:2).
- the amount of vector genomes which could be detected in the lungs (3.8 ⁇ 10 5 ⁇ 1.9 ⁇ 10 5 vg/100 ng total DNA) was about 6-100 times higher than the amount of vector genomes which was demonstrated in another organ ( FIG. 5A ).
- the vector distribution of wild-type AAV2, the control peptide CVGSPCG and the lung-specific peptide ESGHGYF was measured 14 days after intravenous administration of 5 ⁇ 10 10 gp/mouse, i.e., at the time when the expression of the transgene was determined (see above).
- the genomes provided by wild-type AAV2 vectors were mainly recovered from the liver and spleen, and the genomes of vectors which had the control peptide were obtained largely from the spleen.
- the amount of vectors detected in the lung which showed the peptide ESGHGYF (SEQ ID NO: 2) was about 250-fold higher than in other organs, and up to 500-fold higher than in lungs which were injected with a wild-type vector or a control capsid vector ( FIG. 5B ).
- the same distribution values between the organs were found 28 days after vector administration.
- the direct comparison between the three vector capsid variants for the quantities of genomes found is shown in FIG. 5C for the three tissues in which relevant amounts of vector DNA were detected.
- FIG. 5C for the three tissues in which relevant amounts of vector DNA were detected.
- Immunohistochemistry was used to visualize the expression of the transgene at the cellular level in the lung, as well as in a control organ, 14 days after the intravenous administration of the rAAV-GFP vector having the peptide ESGHGYF (SEQ ID NO:2) and/or the wild-type AAV capsid as control.
- the lungs of the animals were fixed ex situ with 4% (w/v) paraformaldehyde via the trachea under hydrostatic pressure of 20 cm of water for 20 minutes, followed by 24 hours of immersion in the same fixative.
- the lung tissues were embedded in paraffin. Sections with a thickness of 2 ⁇ m were removed from wax, rehydrated and used for immunohistochemistry.
- mice injected with rAAV-ESGHGYF SEQ ID NO:9
- a microscopic examination showed intensive staining of the endothelial cells over the entire pulmonary micro-vasculature and to a slightly lesser extent in the large pulmonary vessels (data not shown).
- pulmonary tissue of mice which was injected with wild-type AAV2 vector showed no staining.
- the liver was analyzed as a control organ (a tissue which is known to frequently demonstrate high expression of a transgene) after injection of wild-type AAV2 vector.
- hepatocyte staining was observed after administration of wild-type rAAV2 vector; but no staining was observed after administration of rAAV2-ESGHGYF (SEQ ID NO:9) vector.
- the endothelial lineage of pulmonary cells transduced with the vectors was confirmed by CD31 staining, wherein the pattern obtained by the GFP staining was confirmed in serial sections of the lungs of mice injected with rAAV2-ESGHGYF (SEQ ID NO:9) (data not shown).
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Abstract
Description
-
- (a) the amino acid sequence of SEQ ID NO: 9;
- (b) an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 9; or
- (c) a fragment of one of the amino acid sequences defined in (a) or (b).
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Nat Biotechnol 21, 1040-1046 - [27] Water Kamp, et al., 2006 J Gene Med 8, 1307-1319
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Gene Ther 6, 973-985 - [30] McCarty et al., 2001, Gene Ther 8, 1248-1254
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Exp Hematol 35, 1766-1776 - [32] Rohr et al., 2005 J Virol Methods 127, 40-45
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EP3536794A3 (en) | 2019-11-20 |
JP6560212B2 (en) | 2019-08-14 |
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US20160175389A1 (en) | 2016-06-23 |
JP2019214579A (en) | 2019-12-19 |
US20230181683A1 (en) | 2023-06-15 |
JP2016527298A (en) | 2016-09-08 |
EP3030664A1 (en) | 2016-06-15 |
JP6768895B2 (en) | 2020-10-14 |
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